Gossypol Repressing The Expression Of Connexin43 In Sertoli Cells

The contraceptive effect of gossypol in man had been studied for 30 years. The mechanism and toxicology of gossypol and its allied compound had been widely studied. However, the definite mechanism was still unclear. The study of the reproductive toxicology of gossypol was a very important work in andrology.Sertoli cells were the unique somatocytes in seminiferous of testis. It provided mechanical and nutritional support and played a key role in spermatogenesis. Alterations in Sertoli cell function may lead to a germ cell loss and disruption of the seminiferous epithelium and in turn will lead to impaired spermatogenesis.Gap junctions were composed of intercellular pores that allow the passage of small molecules between adjacent cells. The half channel consisted of a hexamer of specific proteins that belong to the gene family of Connexin. Cx43 was the most important Connexin in the testis, which was localized between Sertoli cells, between Leydig cells, and between Sertoli and germ cells.It was reported that some toxicants such as lindane and cadmium could reduce the connexins in Sertoli cell and block the intercellular communication between cultured human and rat cells. Alterations in gap junctions may be involved in the impairment of spermatogenesis and the pathogenesis of neoplastic seminoma proliferation and then may disrupt the control of germ cell proliferation by Sertoli cells. We presume that the mechanism of suppressing spermatogenesis of gossypol is in the same way.The present study was to investigate the effects of gossypol on Sertoli cell, detect the GJIC on Sertoli cell and approach the mechanism of the Cx43 depression. This would be a significant work in the anti-fertility research of gossypol and would offer a new idea for the studies of the other toxicants. In preliminary experiments, a Sertoli cell line, TM4, was treated with different concentrations of gossypol, 1.25, 2.5, 5 and 10μmol/L for 6, 12, 24 and 48 hours. Cell viability was assessed with CCK-8 assay. GJIC in cells was determined using the scrape loading and dye transfer (SLDT) assay; the expression of Cx43 was detected by RT-PCR, immunofluorescence and Western blot analysis. In the second experiment, we improved the method for Sertoli cells separation from mouse testis in order to obtain more pure cells. Cells were identified by Feulgen staining and FasL detection. RT-PCR was performed to detecte the expression of Cx43. In the third experiment, adult male mice were treated with 15 mg/kg/d gossypol for 4, 8 and 12 weeks. The morphology of the seminiferous epithelium was observed and Cx43 was analyzed by immunohistochemistry.The SLDT assay showed that GJIC between adjacent cells was significantly decreased by gossypol. TM4 cells were incubated with DMSO for 48h or 5μmol/L of gossypol for 6h, 12h, 24h and 48h, respectively. The decrease in Cx43 mRNA occurred as early as 6h after the treatment of gossypol and the effect continued to 48h (P<0.05). Western blot analysis showed that the expression of connexin43 protein was gradually decreased with increasing concentrations when cells were incubated with 1.25, 2.5, 5, or 10μmol/L gossypol for 24 h (P<0.05). The expression of Cx43 was gradually decreased with the increasing concentrations of gossypol, and the effect occurred as early as 6h after the treatment and continued until 48h. The cells were treated with 1.25, 2.5 and 10μmol/L of gossypol for 24 h. The expression of Cx43 was detected by immunofluorescent assay. The intensity of Cx43 immunostaining was dramatically decreased at all doses used (P<0.05). In the second experiment using the combination enzyme digestion method, the purity of Sertoli cells reached above 85%. The RT-PCR assay showed gossypol significantly decreased Cx43 mRNA. In the third experiment, mice were treated with 15 mg/kg/d gossypol, the morphology of the seminiferous epithelium was observed and immunohistochemistry showed that the expression of Cx43 was decreased as early as 8weeks after the treatment and continued until 12weeks.In summary, Cx43 composed gap junctions fundamentally, which were obviously presented between adjacent Sertoli cells. Gossypol could repress the GJIC by decreased the expression of Cx43 and impair the function of Sertoli cells. The seminiferous tubules of mice didn't show any disorder under the treatment of low dose of gossypol. The results suggested the reversibility of the contraceptive effect of gossypol. However, gossypol could repress the expression of Cx43 in Sertoli cells, which is one of the mechanisms of the reproductive toxicology of gossypol.